Discovery and engineering systems for multi-enzyme catalysis

Reprogramming and rewiring biological systems by introducing new functionalities offers great promise for the design of cells for the production of new chemicals. In this lecture I will discuss and show examples of our efforts on engineering metabolic pathways and other complex properties into microbial cells and for in vitro biomanufacturing. In this presentation I will discuss our efforts in i) discovery and characterization of biosynthetic enzymes for the production of natural products and ii) design and engineering of self-assembling protein systems for in vivo and in vitro multi-enzyme biocatalysis. In a first example, I will describe our efforts on characterizing and accessing the natural product portfolio of Basidiomycete fungi with goal of enabling the discovery of new enzyme activities and bioactive compounds. using genomics driven approaches together with heterologous expression and biochemical characterization of enzymes and biosynthetic pathways. Basidiomycota and Ascomycota represent the major phyla of the fungal kingdom. Only a fraction of this diversity has been described and an even smaller fraction of the known species has been characterized in more detail and/or exploited by humans. Ascomycota have so far received the greatest attention while Basidiomycota remain greatly understudied despite their importance for carbon recycling, ecosystem functioning and medicinal properties. This group of fungi holds great promise for the discovery of novel biosynthetic pathways and biocatalysts, especially enzymes for redox catalysis [1]. Homology-based analysis of fungal genomes suggests that the secondary metabolome of Basidiomycota differs significantly from those of other prolific microbial natural products producers [2]. Leveraging genomic information, we have identified and characterized a large number of different sesquiterpene synthases from Basidiomycota. Many of the sesquiterpene synthases are located in associated biosynthetic gene clusters which we have begun to characterize. More recently, we have sequenced additional Basidiomycota genomes that give us access to new types of sesquiterpene scaffolds and clustered biosynthetic enzymes. In a second example, I will discuss our efforts on engineering enzyme co-localization systems for metabolic engineering and biocatalysis. Cells operate a multitude of enzyme cascade reactions simultaneously with high efficiency, while at the same time controlling metabolic fluxes, preventing the build-up of toxic intermediates and directing metabolites to the correct enzymatic pathways [3]. Key to the optimal function of cellular metabolic networks is the spatial organization and temporal control of these cascades. The same design principles for spatial organization of metabolic enzymes may be adapted to engineer more efficient metabolic pathways and create robust and highly efficient cell-free orthogonal biocatalytic cascade reactions that operate concurrently in one pot. We have engineered protein nano-compartments into E. coli and have shown that multiple heterologous cargo proteins can be targeted into these compartments [4, 5]. More recently, we have designed robust self-assembling protein scaffolds that enable co-localization of multiple cargo proteins and enzymes on these protein architectures. Recombinant scaffold building block and cargo proteins can be readily produced for the formation of self-assembled protein systems. [1] C. Schmidt-Dannert (2016) Biocatalytic portfolio of Basidiomycota. Curr. Opin. Chem. Biol. 31:40-49. [2] C. Schmidt-Dannert (2015) Biosynthesis of terpenoid natural products in fungi. Adv. Biochem Eng. Biotechnol. 8:26-8. [3] C. Schmidt-Dannert, F. Lopez-Gallego (2016) A roadmap for biocatalysis – functional and spatial orchestration of enzyme cascades. Microb. Biotechnol. 9:601-609. [4] M. Held, A. Kolb, S. Perdue, S. Hsu, S. Bloch, M.B. Quin, C. Schmidt-Dannert (2016) Engineering formation of multiple recombinant Eut protein nanocompartments in E. coli. SciReports, 6:24359. [5] M.B. Quin, S.A. Perdue, S.Y. Hsu, C. Schmidt-Dannert (2016) Encapsulation of multiple cargo proteins within recombinant Eut nanocompartments. Appl. Microbiol. Biotechnol. 100:9187-9700

Numerical treatment of imprecise random fields in non-linear solid mechanics

The quantification and propagation of mixed uncertain material parameters in the context of solid mechanical finite element simulations is studied. While aleatory uncertainties appear in terms of spatial varying parameters, i.e. random fields, the epistemic character is induced by a lack of knowledge regarding the correlation length, which is therefore described by interval values. The concept and description of the resulting imprecise random fields is introduced in detail. The challenges occurring from interval valued correlation lengths are clarified. These include mainly the stochastic dimension, which can become very high under some circumstances, as well as the comparability of different correlation length scenarios with regard to the underlying truncation error of the applied Karhunen-Loève expansion. Additionally, the computation time can increase drastically, if the straightforward and robust double loop approach is applied. Sparse stochastic collocation method and sparse polynomial chaos expansion are studied to reduce the number of required sample evaluations, i.e. the computational cost. To keep the stochastic dimension as low as possible, the random fields are described by Karhunen-Loève expansion, using a modified exponential correlation kernel, which is advantageous in terms of a fast convergence while providing an analytic solution. Still, for small correlation lengths, the investigated approaches are limited by the curse of dimensionality. Furthermore, they turn out to be not suited for non-linear material models. As a straightforward alternative, a decoupled interpolation approach is proposed, offering a practical engineering estimate. For this purpose, the uncertain quantities only need to be propagated as a random variable and deterministically in terms of the mean values. From these results, the so-called absolutely no idea probability box (ani-p-box) can be obtained, bounding the results of the interval valued correlation length being between zero and infinity. The idea is, to interpolate the result of any arbitrary correlation length within this ani-p-box, exploiting prior knowledge about the statistical behaviour of the input random field corresponding to the correlation length. The new approach is studied for one- and two-dimensional random fields. Furthermore, linear and non-linear finite element models are used in terms of linear-elastic or elasto-plastic material laws, the latter including linear hardening. It appears that the approach only works satisfyingly for sufficiently smooth responses but an improvement by considering also higher order statistics is motivated for future research.DFG/SPP 1886/NA330/12-1/E

Rusty Hungary: New Insights in Brownfield Research

Spatial development and planning in Hungary bear the marks of the post-socialist transformation. This is also observed in the handling of brownfields which, in the wake of political changes, increased in large numbers. In recent years a lot has happened, but we still know far too little about these areas. The paper focuses on the assessment of the extent of brownfields, their properties and regeneration in Hungary. The survey was based on collection of existing and accessible data at national and regional level, complemented by two personal databases. The study extends beyond the spatial dimension of brownfield sites. The results also emphasize further contexts of brownfield management that are not easily perceptible

Influence of suspension viscosity on Brownian relaxation of filler particles

Brownian relaxation caused by Brownian movement of particles in suspensions can macroscopically be probed by small-amplitude oscillatory shear experiments. Phenomenological considerations suggest a direct proportionality between suspension viscosity and Brownian relaxation times. To verify this relation experimentally, a set of nanocomposite suspensions with viscosities varying over five decades is presented. The suspensions are chosen in a way to ensure that particle-particle interactions and average particle-particle distances are identical so that they can be used as a model system to study the mere influence of suspension viscosity on Brownian relaxation. The suggested linear relationship between suspension viscosity and Brownian relaxation time can be confirmed. Moreover, a verification of a recently introduced characteristic timescale for Brownian relaxation is presented